Giorgio Dieci

Eukaryotic snoRNAs: a paradigm for gene expression flexibility.

Small nucleolar RNAs (snoRNAs) are one of the most ancient and numerous families of non-protein-coding RNAs (ncRNAs). The main function of snoRNAs - to guide site-specific rRNA modification - is the same in Archaea and all eukaryotic lineages. In contrast, as revealed by recent genomic and RNomic studies, their genomic organization and expression strategies are the most varied. Seemingly snoRNA coding units have adopted, in the course of evolution, all the possible ways of being transcribed, thus providing a unique paradigm of gene expression flexibility. By focusing on representative fungal, plant and animal genomes, we review here all the documented types of snoRNA gene organization and expression, and we provide a comprehensive account of snoRNA expressional freedom by precisely estimating the frequency, in each genome, of each type of genomic organization. We finally discuss the relevance of snoRNA genomic studies for our general understanding of ncRNA family evolution and expression in eukaryotes.

17A, a novel non-coding RNA, regulates GABA B alternative splicing and signaling in response to inflammatory stimuli and in Alzheimer disease

Alternative splicing is a central component of human brain complexity; nonetheless, its regulatory mechanisms are still largely unclear. In this work, we describe a novel non-coding (nc) RNA (named 17A) RNA polymerase (pol) III-dependent embedded in the human G-protein-coupled receptor 51 gene (GPR51, GABA B2 receptor). The stable expression of 17A in SHSY5Y neuroblastoma cells induces the synthesis of an alternative splicing isoform that abolish GABA B2 intracellular signaling (i.e., inhibition of cAMP accumulation and activation of K(+) channels). Indeed, 17A is expressed in human brain, and we report that it is upregulated in cerebral tissues derived from Alzheimer disease patients. We demonstrate that 17A expression in neuroblastoma cells enhances the secretion of amyloid β peptide (Aβ) and the Aβ x-42/Αβ x-40 peptide ratio and that its synthesis is induced in response to inflammatory stimuli. These data correlate, for the first time, the activity of a novel pol III-dependent ncRNA to alternative splicing events and, possibly, to neurodegeneration induced by abnormal GABA B function. We anticipate that further analysis of pol III-dependent regulation of alternative splicing will disclose novel regulatory pathways associated to brain physiology and/or pathology.

RNA polymerase III transcription control elements: Themes and variations

Eukaryotic genomes are punctuated by a multitude of tiny genetic elements, that share the property of being recognized and transcribed by the RNA polymerase (Pol) III machinery to produce a variety of small, abundant non-protein-coding (nc) RNAs (tRNAs, 5S rRNA, U6 snRNA and many others). The highly selective, efficient and localized action of Pol III at its minute genomic targets is made possible by a handful of cis-acting regulatory elements, located within the transcribed region (where they are bound by the multisubunit assembly factor TFIIIC) and/or upstream of the transcription start site. Most of them participate directly or indirectly in the ultimate recruitment of TFIIIB, a key multiprotein initiation factor able to direct, once assembled, multiple transcription cycles by Pol III. But the peculiar efficiency and selectivity of Pol III transcription also depends on its ability to recognize very simple and precisely positioned termination signals. Studies in the last few years have significantly expanded the set of known Pol III-associated loci in genomes and, concomitantly, have revealed unexpected features of Pol III cis-regulatory elements in terms of variety, function, genomic location and potential contribution to transcriptome complexity. Here we review, in a historical perspective, well established and newly acquired knowledge about Pol III transcription control elements, with the aim of providing a useful reference for future studies of the Pol III system, which we anticipate will be numerous and intriguing for years to come.

New small nuclear RNA gene-like transcriptional units as sources of regulatory transcripts

By means of a computer search for upstream promoter elements (distal sequence element and proximal sequence element) typical of small nuclear RNA genes, we have identified in the human genome a number of previously unrecognized, putative transcription units whose predicted products are novel noncoding RNAs with homology to protein-coding genes. By elucidating the function of one of them, we provide evidence for the existence of a sense/antisense-based gene-regulation network where part of the polymerase III transcriptome could control its polymerase II counterpart.

Detours and shortcuts to transcription reinitiation

Gene transcription is repetitive, enabling the synthesis of multiple copies of identical RNA molecules from the same template. The cyclic process of RNA synthesis from active genes, referred to as transcription reinitiation, contributes significantly to the level of RNAs in living cells. Contrary to the perception that multiple transcription cycles are a mere iteration of mechanistically identical steps, a large body of evidence indicates that, in most transcription systems, reinitiation involves highly specific and regulated pathways. These pathways influence the availability for reinitiation of template DNA and/or transcription proteins, and represent an important yet poorly characterized aspect of gene regulation.

Visualizing RNA extrusion and DNA wrapping in transcription elongation complexes of bacterial and eukaryotic RNA polymerases.

Transcription ternary complexes of Escherichia coli RNA polymerase and yeast RNA polymerase III have been analyzed by atomic force microscopy. Using the method of nucleotide omission and different DNA templates, E.coli RNAP has been stalled at position +24, +70 and +379 and RNAP III at position +377 from the starting site. Conformational analysis of E.coli RNAP elongation complexes reveals an average DNA compaction of 22nm and a DNA deformation compatible with approximately 180 degrees DNA wrapping against the enzyme. The extent of protein-DNA interaction attributed to wrapping, however, is less than that of corresponding open promoter complexes. DNA wrapping was also observed for RNAP III elongation complexes, which showed a DNA compaction of 30nm. When the RNA polymerases were stalled far from the promoter (+379 and +377), the growing RNA transcript was often visible and it was prevalently seen exiting from the enzyme on the opposite side relative to the smallest angle subtended by the upstream and downstream DNA arms. Surprisingly, we found that many complexes had a second RNAP, not involved in transcription, bound to the growing RNA of a ternary complex. DNA wrapping in the elongation complex suggests a possible mechanism by which the polymerase may overcome the physical barrier to transcription imposed by the nucleosomes.

An intronic ncRNA-dependent regulation of SORL1 expression affecting Aβ formation is upregulated in post-mortem Alzheimer's disease brain samples.

SUMMARY Recent studies indicated that sortilin-related receptor 1 (SORL1) is a risk gene for late-onset Alzheimers disease (AD), although its role in the aetiology and/or progression of this disorder is not fully understood. Here, we report the finding of a non-coding (nc) RNA (hereafter referred to as 51A) that maps in antisense configuration to intron 1 of the SORL1 gene. 51A expression drives a splicing shift of SORL1 from the synthesis of the canonical long protein variant A to an alternatively spliced protein form. This process, resulting in a decreased synthesis of SORL1 variant A, is associated with impaired processing of amyloid precursor protein (APP), leading to increased Aβ formation. Interestingly, we found that 51A is expressed in human brains, being frequently upregulated in cerebral cortices from individuals with Alzheimers disease. Altogether, these findings document a novel ncRNA-dependent regulatory pathway that might have relevant implications in neurodegeneration.

An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III‐transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.—Castelnuovo, M., Massone, S., Tasso, R., Fiorino, G., Gatti, M., Robello, M., Gatta, E., Berger, A., Strub, K., Florio, T., Dieci, G., Cancedda, R., Pagano, A. An Alu‐like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells. FASEB J. 24, 4033–4046 (2010). www.fasebj.org

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNAs.

The telomere-binding protein Tbf1 demarcates snoRNA gene promoters in Saccharomyces cerevisiae.

Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA biogenesis, yet factors controlling their expression are unknown. We found that the majority of Saccharomyces snoRNA promoters display an aRCCCTaa sequence motif at the upstream border of a TATA-containing nucleosome-free region. Genome-wide ChIP-seq analysis showed that these motifs are bound by Tbf1, a telomere-binding protein known to recognize mammalian-like T(2)AG(3) repeats at subtelomeric regions. Tbf1 has over 100 additional promoter targets, including several other genes involved in ribosome biogenesis and the TBF1 gene itself. Tbf1 is required for full snoRNA expression, yet it does not influence nucleosome positioning at snoRNA promoters. In contrast, Tbf1 contributes to nucleosome exclusion at non-snoRNA promoters, where it selectively colocalizes with the Tbf1-interacting zinc-finger proteins Vid22 and Ygr071c. Our data show that, besides the ribosomal protein gene regulator Rap1, a second telomere-binding protein also functions as a transcriptional regulator linked to yeast ribosome biogenesis.